Association of β1 and β2-adrenoceptor Polymorphisms With the Demand of Inotropic Catecholamine Support Following Coronary Artery Bypass Grafting in Iranian Population

Background: Hemodynamic instability is a common complication in the rst hours following cardiac surgery and inotropic catecholamine support is an acceptable treatment strategy for its management. β 1 and β 2 -adrenoceptors (β 1 and β 2 AR) are mediated the positive inotropic and chronotropic responses of the heart to catecholamines. Previous evidence has suggested an association between β 1 and β 2 AR polymorphisms and cardiac response and change in receptor signaling. The aim of this study was evaluating the relationship between β 1 and β 2 AR polymorphisms with demand of catecholamine inotropic support among coronary artery bypass grafting (CABG) patients. Methods: One hundred ninety-eight consecutive patients who underwent CABG with cardiopulmonary bypass were included in this study. We assessed hemodynamic parameters, dose and duration of inotropic support according to β 1 and β 2 AR genotypes in post-operative period. DNA genotyping were assessed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR genotyping results were conrmed by direct DNA sequencing. Results: The our results indicated that patients carrying one or two alleles of the Arg389-β 1 AR variant required signicantly shorter inotropic support time compared with patients homozygous for the Gly389-β 1 AR (p=0.003). Finally, neither β 1 AR polymorphisms nor Arg16Gly-β 2 AR polymorphism are associated with catecholamines-induced hemodynamic effects. Conclusion: These ndings suggest that genetic variability in the β 1 and β 2 AR polymorphisms may not be a major determinant of cardiac responses to catecholamine treatment in Iranian population. However, larger scale studies with different ethnicities are needed for conrmation. categorical variables. Differences between means were compared using the one-way analysis of variance (ANOVA). Post-hoc analysis was performed when ANOVA was indicated signicance. The Hardy-Weinberg equilibrium also was tested by the chi-square test to determine the frequency distribution of genotypes. P<0.05 was considered as statistical differences in each test. 0.003). inotrope infusion inotropic 20.5 Arg389Arg-β conclude, our results demonstrated that in patients undergoing CABG surgery with CPB who carrying one or two alleles of the Arg389-β 1 AR, the time of inotrope support to achieve positive inotropic response was signicantly shortest than in Gly389-β 1 AR homozygotes. Furthermore, data from this study revealed that no signicant association between patient’s hemodynamic response to inotropic support after CABG surgery and three polymorphisms studied have been observed in Iranian population. However, to provide stronger evidence further genetic analysis with different and larger populations is required.


Background
Hemodynamic disturbances and impaired cardiac functions are major problems and cause of death following coronary artery bypass grafting (CABG) surgery with cardiopulmonary bypass (CPB) that is secondary to myocardial hypoxia or ischemia [1]. The condition is manifested by cardinal signs such as hypotension, peripheral vasoconstriction and oliguria. The mechanism of post-operative hypotension is incompletely understood. It is believed that systemic in ammation and vasoplegia that occur in patients following major cardiac surgery manifest as systemic arterial vasodilation and profound hypotension with a high cardiac index and a low systemic vascular resistance [2]. Inotropic support by catecholamines are the cornerstone treatment of hypotensive post-CABG states to improve blood pressure and organ perfusion via increasing cardiac output with effect on beta and alfa adrenergic receptors. However, in many patients who experience post-CABG depressed myocardial function, inadequate and weak response to catecholamines is observed and identi cation of patients who will adequately respond to inotrope therapy remains a challenge [3]. β 1 -adrenergic pathway through positive inotropic and chronotropic effects, play a key role in the regulation of heart rate and contractility and is responsive to the effects of circulating catecholamines [4,5]. In addition, β 2 -adrenoceptors (β 2 ARs) are also expressed on the myocardium, atria of the heart and in vascular smooth muscle beds. β 2 ARs are allowed for calcium in ux in response to cardiac sympathetic nerve activity, leading to positive inotropic and chronotropic effects [6. 7]. Recent in vivo and in vitro studies have shown multiple genetic variants in β 1 and β 2 ARs [8,9]. This implies that variations in the β 1 and β 2 AR gene might be explain some variability observed in response of patients CABG surgery to inotropic support therapy.
Enhanced left ventricular ejection fraction and also greater stroke volume, cardiac output and mean arterial pressure has been observed in healthy subjects homozygous for the Gly16-β 2 AR allele [10,11]. Additionally, previous studies (Bruck et al., 2005, La Rosée et al., 2004 have demonstrated a higher heart rate and/or contractility among individuals homozygous for the Arg389-β 1 AR variant compared with other codon 389 genotype carriers [12,13]. A study done by Leineweber et al., 2007, in sample of German population showed that less post-surgical inotropic support is required in patients undergoing CABG who were preoperatively chronically treated with metoprolol and homozygous for the Arg389-β 1 AR, than those with one or two Gly389-β 1 AR alleles [14]. Over-responsiveness to propranolol has been reported in Iranian population that may be associated with polymorphism of β-adrenoceptors [15]. Furthermore, it was observed that Arg16Gly-β 2 AR genotype may have protective effect for hypertension in comparison with Arg16Arg-β 2 AR variant in Iranian population [16]. Therefore, the present study was designed to investigate whether the hemodynamic responses to inotropic support by epinephrine in Iranian patients with hemodynamic instability following CABG surgery with CPB are in uenced by β 1 and β 2 AR polymorphic variations.

Study population
This was a prospective study included 198 patients attending Mazandaran Heart Center, Sari, Iran between April and September 2018. The patients aged 18 years or older and indicated for elective CABG surgery with CPB that received inotropic support only by epinephrine after weaning from CPB or in the rst 12 hours after post-operative ICU admission were included in this study. The patients who needed a pacemaker, intra-aortic balloon pump after weaning from CPB, those who also had post-operative major bleeding (> 200 ml/h rst 6 h after surgery), septic shock, and preoperative end-stage renal disease or post-operative severe renal failure (creatinine clearance < 30 ml/min/1.73 m 2 ) requiring temporary hemodialysis were also excluded from the study. The study protocol was approved by the ethics committee of Mazandaran University of Medical Sciences.

Data collection and clinical assessment
Pre and post-operative demographics, medical history and operative details were extracted from patient's medical records. Preoperatively, the usual doses of cardiac medications were prescribed for all participants and none received inotropic support.
A systolic blood pressure (SBP) < 90 mmHg or mean arterial pressure (MAP) < 65 mmHg was considered as the post-operative hypotension that required inotropic support. Inotropic support by epinephrine was started at the rate of 10 ng/kg/min and titrated against blood pressure to achieve hemodynamic target of mean arterial pressure between 70 and 90 mmHg.
At the time when the inotrope infusion was initiated, systolic and diastolic blood pressure (DBP), mean arterial pressure, heart rate (HR), central venous pressure (CVP) and urine output were recorded. During the inotrope infusion, these parameters were frequently monitored. In addition, the required dose and total duration of epinephrine infusion were recorded. In order to maintain adequate circulating volume and electrolyte balance during inotrope administration, all patients received intravenous isotonic uids.

DNA extraction
Five milliliters of peripheral venous blood samples were taken in tubes containing ethylene diamine tetraacetic acid (EDTA) and preserved at -80 °C for isolation of DNA. Genomic DNA was extracted using a column-based DNA isolation kit (Denazist Asia Co., Mashhad, Iran) according to the manufacturer's instructions. The concentration and purity of the extracted DNA were measured using a Nanodrop spectrophotometer (Biochrom WPA Biowave II + UV/visible spectrophotometer, Cambridge, UK).

Genotyping using PCR-RFLP method
A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed to determine the β 1 AR gene polymorphisms (codon 389, nucleotide 1165 and codon 49, nucleotide 145) and β 2 AR gene polymorphism (codon 16, nucleotide 46). PCR was performed using 200 ng of template DNA, 10 μl Taq DNA polymerase 2x master mix RED (Ampliqon A/S, Odense, Denmark) containing 150 mM Tris-HCl pH 8.5, 40 mM (NH 4 ) 2 SO 4 , 3 mM MgCl 2 , 0.2% Tween 20, 0.4 mM of each dNTP and 0.2 U/μl Ampliqon Taq DNA polymerase, along with 10 pmol of speci c primers, and 7 µl nuclease-free water. The PCR condition, sequence of primers and expected product lengths are listed in Table 1. The PCR products were restriction-digested with 10 U/µl of BcgI (β 1 AR codon 389), EcoO109I (β 1 AR codon 49) (Fermentas; Thermo Fisher Scienti c, Inc., Waltham, MA, USA) at 37˚C for 3 hours and BsrDI (β 2 AR codon 16) (Bio Lab; New England) at 55˚C for 3 hours. Digested fragments were analyzed by electrophoretic separation on 2% agarose gel containing DNA green uorescent dye (DenaZist, Mashhad, Iran) (Fig. 1). The staffs of the ICU and the investigators were blinded to the genotypes of the patients. The genotypes determined by PCR-RFLP were further con rmed by direct sequencing (Fig. 1). Genotyping was performed without knowing the population study status. Ten percent of random samples were tested by Sanger sequencing with forward and reverse primers by ABI 3130XL.

Statistical analysis
The data were analyzed by SPSS Statistics software, version 22 (SPSS Inc., Chicago, Illinois, USA). Continuous variables were quoted as mean ± standard deviation (SD) and the results on the categorical measurements were expressed as numbers and percent. Chi-square test and Fisher's exact test were used for categorical variables. Differences between means were compared using the one-way analysis of variance (ANOVA). Post-hoc analysis was performed when ANOVA was indicated signi cance. The Hardy-Weinberg equilibrium also was tested by the chi-square test to determine the frequency distribution of genotypes. P<0.05 was considered as statistical differences in each test.
Baseline demographics, operative details and post-operative clinical information of patients according to by β 1 and β 2 AR genotypes are listed in Tables 2 and 3, respectively. Baseline characteristics were similar in all variants ( Table 2). As illustrated in Table 3, Operative details and post-operative data were similar between the study population with different polymorphisms. However, regarding the Arg16Gly-β2AR polymorphism, post-operative left ventricular ejection fraction (LVEF) was signi cantly higher in patients with the Arg16Arg genotype with 55 ± 5.5% versus 48.5 ± 9.2% in patients with Arg16Gly and 51.1 ± 7% in Gly16Gly carriers (p = 0.028). There was no statistically signi cant difference in post-operative LVEF between the two β 1 AR polymorphisms.    Table 3 Operative details and post-operative data of patients according to Arg389Gly-β 1 AR, Ser49Gly-β 1 AR and Arg16Gly-β 2   Hemodynamic variables and changes in hemodynamic parameters as compared to baseline after the initiation of epinephrine infusion are presented in Table 4 and Fig. 2. The results showed that increases in SBP, DBP, MAP and HR were similar after inotropic support by epinephrine and there were no signi cant differences between the three polymorphisms studied. Similar results were also obtained for decrease in CVP according to β 1 and β 2 AR genotypes. Although Gly389Gly-β 1 AR and Gly16Gly-β 2 AR homozygous and Ser49Gly-β 1 AR heterozygous patients had a higher changes in HR compared to other variants, no signi cant differences were found between the three βAR polymorphisms. Furthermore, the increase in SBP and MAP and decrease in CVP were, in patients heterozygous for the Ser49Gly-β 1 AR variant, higher than patients with other variants. However, these changes were small and not statistically signi cant (Table 4, Fig. 2). As can be seen from Fig. 2, in patients carrying one or two Arg alleles at codon 389, the duration of inotropic support after CABG surgery with CPB were signi cantly shorter (P < 0.05) than patients homozygous for the Gly389-β 1 AR. There was a trend towards lower dose of epinephrine required to achieve the hemodynamic goal in patients homozygous for the Arg389-β 1 AR, Ser49-β 1 AR and Gly16-β 2 AR variants compare to other genotypes, although this difference just failed to reach statistical signi cance.

Discussion
Poor cardiac function and post-operative hypotension during the early hours after CABG surgery with CBP is associated with a number of serious complications including vasospasm, generalized in ammatory response, organ dysfunction and an increased risk of death [17]. Epinephrine is an effective initial inotropic agent that widely used in patients with hypotension following cardiac surgery and clearly improves cardiac output and myocardial performance [18]. However, among post-cardiac surgery patients different responses to inotropic agents were observed. Beta-adrenoceptors mediate the physiological effects of catecholamines and also these receptors are polymorphic. Hence, inter-individual differences in genetic composition could lead to various drug responses and it could give rise to innovating effective treatment approaches. Although previous studies (Leineweber et al., 2007, Dhein et al., 2017 have proven the signi cant association of β 1 AR gene and its polymorphisms in cardiovascular diseases and their impact on responses to βAR agonist treatment, still its role as therapeutic determinant remains unclear [14,19]. According to our knowledge, there have been neither previous investigations of the genetic basis of hemodynamic effects of positive inotropic agents in patients undergoing CABG surgery with CPB in Iranian population. Results of our study suggest lack of any association between the three polymorphisms studied and patient's hemodynamic response to inotropic support after CABG surgery with CPB. In addition, patients carrying one or two Arg389-β 1 AR alleles needed signi cantly less inotropic support time compared to Gly389-β 1 AR homozygotes. Evidence suggests that mutant Arg389Arg-β 1 AR is a known genetic risk factor for cardiovascular diseases [8,20]. In the present study, we found 41 (20.7%) patients were carrying mutant Arg389Arg-β 1 AR genotype, which is lower than the reported frequency in other white populations [4,19,21]. Regarding the genotypes found in our population, the homozygous Gly49Gly-β 1 AR variant was observed only in one patient. The Gly49Gly variant is extremely rare and its lower frequency was also reported in previous studies within various ethnic populations (22)(23)(24)(25). Furthermore, in good accordance with our data, Gly16-β 2 AR and Gly16Gly-β 2 AR were reported as the most prominent alleles and genotypes among different ethnic populations (White and Black Americans, Chinese and Egyptian populations) [26,27].
The Gly389-β 1 AR has a 3-4-fold fewer isoprenaline stimulation of the adenylyl cyclase than the Arg389-β 1 AR variant [12]. Accordingly, patients carrying the Arg389-β 1 AR allele would be expected more responsive to inotropic therapy and also need lower doses of catecholamine than others. This is in accordance with the ndings of a study by Dhein et al., 2017, that also reported more norepinephrine requirements in patients with Gly389 variant of the β 1 AR compared to those carrying one or two Arg389-β 1 AR alleles [19]. In addition, Leineweber et al., 2007, also found that the Gly389Gly-β 1 AR variant was signi cantly associated with higher required dose of epinephrine to reach stable and comparable hemodynamic response [14]. However, the results of our study were in disagreement with data obtained from above-mentioned studies. The present data showed that the dose of epinephrine needed for improvement of cardiac function in patients homozygous for Arg389-β 1 AR was two times higher than for subjects homozygous for Gly389-β 1 AR. On the other hand, patients who carried Gly389Gly-β 1 AR genotype responded two times more to inotropic support therapy in comparison with patients homozygous for Arg at codon 389, although this just failed to reach statistical signi cance (p = 0.703). According to our nding, it may be postulated that there is an Arg389Gly-β 1 AR gene-dose effect.
It was reported that the time of inotropic support was the shortest in patients homozygous for the Arg allele at codon 389, compared to patients homozygous for the Gly389-β 1 AR variant [14]. This is in good accordance to present data. Our results demonstrated that the time of inotropic support to achieve positive inotropic response was signi cantly shortest in Arg389Gly-β 1 AR heterozygous and Arg389Arg-β 1 AR homozygous patients (p = 0.003). Furthermore, comparing with the reported inotrope infusion time in Leineweber et al., 2007 study, the mean inotropic support time was relatively shorter in current study (2.71 vs 20.5 hours in Arg389Arg-β 1 AR and 6 vs 10.5 hours in Gly389Gly-β 1 AR) [14].
Present ndings did not show any statistically signi cant association between hemodynamic measurements (SBP, MAP, CVP, HR) and β 1 AR variants at codon 389 in patients receiving inotropic support (p = 0.0.922, p = 0.807, p = 0.974 and p = 0.274, respectively). Similarly, the in uence of the Arg389Gly-β 1 AR polymorphism on modi ed hemodynamic responses to epinephrine and norepinephrine in young healthy adults was not observed [28,29]. It is worth noting that several studies on the importance of genetic variations of β 1 AR on hemodynamic responses yielded con icting results. In this context, in a study (Bruck et al., 2005) comparing cardiac responses to β 1 AR stimulation by rather β 1 AR selective agonist dobutamine in healthy subjects, improvement of cardiac function was superior in the cardiac contractility and blood pressure in individuals carrying the Arg389Arg-β 1 AR homozygous than the patients carrying the Gly389Gly-β 1 AR variant [12]. Moreover, administration of dobutamine in subjects homozygous for the Arg389-β 1 AR caused signi cantly larger increases in heart rate compared to carriers of one or two alleles of the Gly389-β 1 AR [8,13]. These differences may be postulated that inter-individual variations in required time to improve cardiac function and hemodynamic response to inotropic support after cardiac surgery might be in association with other different genetic mutations and variations of adrenergic system.
According to the results of our study, the Ser49Gly-β 1 AR polymorphism has no signi cant in uence on SBP, MAP, CVP and HR (p = 0.505, p = 0.309, p = 0.859 and p = 0.179, respectively). This is consistent with the ndings Kumar et al., 2008, who found that Ser49Gly-β 1 AR polymorphism did not alter the cardiac response and also systolic and diastolic blood pressures among South Indians [24]. Ser49Ser-β 1 AR was found to be associated with a higher mean HR under resting conditions, which might result in a higher heart rate as we observed for Ser49Ser-β 1 AR genotype carriers, although this just failed to reach statistical signi cance (p = 0.179) (Fig. 2) [30]. In our inotropic support patients with Ser49Ser-β 1 AR variant, a lower dose of epinephrine was required to achieve positive inotropic response than Ser49Gly-β 1 AR genotype. Meanwhile, the Gly49 variant of the β 1 -adrenoceptor gene was shown to be associated with much higher desensitization and faster down-regulation of the β 1 -adrenoceptors [31][32][33]. On the other hand, in current study, patients with the Ser49Ser-β 1 AR variant responded better to the inotropic support than those carrying Gly49-β 1 AR allele; however, there was no signi cant difference between the two variant carriers (p = 0.831).
In vitro studies exhibited that the Gly16-β 2 AR allele enhanced βAR down-regulation [26]. In accordance with this theory, this downregulation would be expected to decrease heart rate and also attenuating the effects of inotropic and chronotropic of catecholamines. This might result in higher required dose of epinephrine in patients carrying one or two Gly16-β 2 AR alleles as observed in our study. By contrast, present ndings demonstrated that increase in heart rate in response to βAR stimulation by epinephrine in Arg16Arg-β 2 AR patients was less than other β 2 AR variants. However, we did not nd any association between the Arg16Gly-β 2 AR polymorphism and increases in heart rate (p = 0.314). This was in line with previous studies performed in young healthy subjects that failed to nd any signi cant genotypedependent differences between homozygous for the Arg16 or Gly16-β 2 AR in male volunteers with increases in heart rate and contractility induced by terbutaline infusion [34,35]. Down-regulation of β 2 AR genotype may occur genotype independently at a similar level among all patients; this would explain a similar response to inotropic support observed in current study.
A previous study (Snapir et al., 2003) has shown that systemic infusions of βAR agonist (epinephrine) are associated with larger vasodilation in subjects homozygous for the Arg16-β 2 AR compared to those carrying one or two Gly16 alleles [28]. Theoretically, this larger vasodilation could be related to lower MAP or SBP in Arg16Arg-β 2 AR homozygotes but our study failed to nd any in uence of the Arg16Gly-β 2 AR polymorphism on hemodynamic parameters (SBP, p = 0.839 and MAP, p = 0.0.669). It might be due to other compensatory cardiovascular re exes which may have overshadowed the agonist activity of catecholamines.
Taken together, chronotropic and inotropic effects of epinephrine were similar across all the different genotypes studied. Findings of the current research and several previous studies have demonstrated variable results [14,19]. The reasons for these differences are not clearly de ned, but may be related to differences in genetic properties of various races.

Limitations
Sample size of this study was relatively limited. It must be recognized that the lack of impact of these polymorphisms on hemodynamic response of post-CABG surgery patients observed in the present study is only hypothesis-generating and further researches with a bigger sample size and different ethnic background are certainly needed to give more reliable data for general population of Iranian patients. Our study demonstrated that the numbers of study's male participants were almost double the number of female patients which indicate that these results may be generalizable and applicable to male patients. As of now, several functionally important SNPs in the human β 1 and β 2 AR coding region have been described. Hence, evaluation of the other SNPs, particularly in the β 2 AR coding region such as Gln27Glu and Thr164Ile polymorphisms would have provided additional important information.

Conclusion
To conclude, our results demonstrated that in patients undergoing CABG surgery with CPB who carrying one or two alleles of the Arg389β 1 AR, the time of inotrope support to achieve positive inotropic response was signi cantly shortest than in Gly389-β 1 AR homozygotes.
Furthermore, data from this study revealed that no signi cant association between patient's hemodynamic response to inotropic support after CABG surgery and three polymorphisms studied have been observed in Iranian population. However, to provide stronger evidence further genetic analysis with different and larger populations is required.  . 1397.1810), Sari, Iran. The funding bodies played no role either in the design of the study, data collection, analysis, interpretation of data, or in the writing of the manuscript.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the author for correspondence upon reasonable request.
Ethics approval and consent to participate The study was approved by the ethics committee of Mazandaran University of Medical Sciences (approval code: IR.MAZUMS.REC.1397.1810). All participants provided written voluntary informed consent prior to the study.

Consent for publication
Not Applicable.